Abstract

BACKGROUND:

MicroRNA (miRNA) are diverse in sequence and have a single known sequence bias: they tend to start with uridine (U).

RESULTS:

Our analyses of fly, worm and mouse miRNA sequence data reveal that the 5'-U is recognized after miRNA production. Only one of the two strands can be assembled into Argonaute protein from a single miRNA/miRNA* molecule: in fly embryo lysate, a 5'-U promotes miRNA loading while decreasing the loading of the miRNA*.

CONCLUSION:

We suggest that recognition of the 5'-U enhances Argonaute loading by a mechanism distinct from its contribution to weakening base pairing at the 5'-end of the prospective miRNA and, as recently proposed in Arabidopsis and in humans, that it improves miRNA precision by excluding incorrectly processed molecules bearing other 5'-nt.

Fly miRNA tend to start with U. Each miRNA or miRNA* isoform derived from a common pre-miRNA was weighted according to its abundance in the pooled deep-sequencing libraries, and the sequence composition analyses for all small RNA from different pre-miRNA that were read at least 100 times in the pooled libraries were weighted equally. Gray, nucleotide frequency at position 1; white, 100 sets of nucleotides randomly selected from nt 1-18 of the miRNA and miRNA* species to assess the overall nucleotide composition of miRNA and miRNA*. Each random set had the same size as the corresponding set of miRNA or miRNA* 5′-nt. P values measure the probability of picking a random set from nt 1-18 with the same nucleotide frequency as the actual set of 5′-nt.

Fly miRNA asymmetry correlates with the identity of the first nucleotide of the small RNA. miRNA/miRNA* duplexes were binned according to their asymmetry: highly asymmetric, miRNA/miRNA* ≥10 in the pooled deep-sequencing libraries; moderately asymmetric, 10 > miRNA/miRNA* ≥ 2; quasisymmetric: miRNA/miRNA* <2; and analyzed as in Figure 1.

Identity of the first miRNA nucleotide affects duplex asymmetry. RNA-induced silencing complex (RISC) loading of miRNA and miRNA* strands was measured after in vitro assembly. Data are reported as means ± standard deviation for three independent replicate experiments. (A) Swapping the terminal uridine:adenosine (U:A) pair of the miR-2a/miR-2a-1* duplex decreased miRNA loading and increased miRNA* loading. (B) The effect of the terminal A:U pair mainly reflects the identity of the first miRNA nucleotide, which affects both miRNA and miRNA* loading (C), whereas the identity of the facing miRNA* nucleotide has no detectable effect (D).

In Drosophila, the identity of the second miRNA nucleotide influences the effect of the first nucleotide. (A) The y-axis shows the significance (Fisher's exact test) of observed covariation between the identity of miRNA nt 1 and nucleotide identity (black) or base-pairing status (gray) along the miRNA and the miRNA* strand. The low values for position 1 in the black curve occur because the identity of miRNA nt 1 correlates strongly with itself. (B and C) Mutating nt 2 in miR-2a (B) or miR-184 (C) changed the order of nt 1 preference for miRNA loading.